Biodiesel, generally classified as mono-alkyl esters of fats and oils, has become more attractive recently because of its environmental benefits. Although biodiesel is at present successfully produced chemically (using e.g. NaOH and/or sodium methoxide as catalyst), there are several associated problems to restrict its development, such as pre-processing of oil due to high contents of free fatty acids, removal of chemical catalyst from ester and glycerol phase and removal of inorganic salts during glycerol recovery.
The disadvantages caused by chemical catalysts are largely prevented by using lipolytic enzymes as the catalysts and in recent years interest has developed in the use of lipases with or without immobilization in transesterification for the production of biodiesel.
Fungal esterases may be used in the enzymatic production of esters, where they may replace catalysts like mineral acid (e.g. sulphuric acid, hydrogen chloride, and chlorosulfonic acid), amphoteric hydroxides of metals of groups I, II, III, and IV, and others. The use of enzymes for ester synthesis has been described in the prior art, in particular enzymes classified in EC 3.1.1 Carboxylic ester hydrolases according to Enzyme Nomenclature (Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, 1992 or later).
WO 88/02775 discloses lipases A and B from Candida antarctica. It states that C. antarctica lipase B (CALB) is more effective for ester synthesis.
Cutinases are lipolytic enzymes capable of hydrolyzing the substrate cutin. Cutinases are known from various fungi (P. E. Kolattukudy in “Lipases”, Ed. B. Borgström and H. L. Brockman, Elsevier 1984, 471-504). The amino acid sequence of a cutinase from Humicola insolens has been published (U.S. Pat. No. 5,827,719).
Many researchers have reported that a high yield of alkyl esters could be reached in the presence of organic solvents, but because of the toxicity and flammability of organic solvents lipase-catalysed alcoholysis in a solvent-free medium is more desirable. Methanolysis catalysed by lipases has been shown to take place in a water-containing system free of organic solvents. In such systems lipases which are less sensitive to methanol is advantageous (Kaieda et al. J. Biosci. Bioeng. 2001, 91:12-15). It is well known that excessive short-chain alcohols such as methanol might inactivate lipase seriously. However, at least three molar equivalents of methanol are required for the complete conversion of the oil to its corresponding methyl ester. Du et al. (Biotechnol. Appl. Biochem. 2003, 38:103-106) studied the effect of molar ratio of oil/methanol comparatively during non-continuous batch and continuous batch operation.
To avoid inactivation of the lipases the methanol concentration has been kept low by step-wise addition of methanol throughout the reaction (Shimada et al. J Mol. Catalysis Enzymatic, 2002, 17:133-142; Xu et al. 2004, Biocat. Biotransform. 22:45-48).
Boutur et al. (J. Biotechnol. 1995, 42:23-33) reported a lipase from Candida deformans which were able to catalyse both alcoholysis of triglyceride (TG) and esterification of free fatty acids (FFA), but not under the same reaction conditions. Under the conditions described by Boutur et al. only the esterification was catalysed.
In order to obtain a more economic production of fatty acid ethyl esters for biodiesel, there is a need for a faster conversion of fats and oils to their corresponding methyl esters and a higher yield in said conversion.